JP6499182B2 - Beta-hairpin peptidomimetics as selective elastase inhibitors - Google Patents

Description

  The present invention provides β-hairpin peptidomimetics that are useful as inhibitors of protease enzymes and have a general disclosure but not specifically disclosed in WO2006 / 087001 A1.

The β-hairpin peptidomimetic of the present invention has the general formula cyclo (Xaa ¹ -Xaa ² -Thr ³ -Xaa ⁴ -Ser ⁵ -Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Xaa ¹² −Xaa ¹³ −)
^{(Xaa 1, Xaa 2, Xaa} 4, Xaa 6, Xaa 7, Xaa 8, Xaa 9, Xaa 10, Xaa 11, Xaa 12 and Xaa ¹³ are the specification and the specific types of which are defined in the appended claims Amino acid residues)
And a pharmaceutically acceptable salt thereof.

  These β-hairpin peptidomimetics are useful as inhibitors of protease enzymes, and are particularly useful as inhibitors of certain serine proteases such as elastase.

  Furthermore, the present invention provides an efficient production method in which these compounds can be obtained in a library format if necessary.

  The β-hairpin peptidomimetic of the present invention exhibits high inhibitory activity against human neutrophil elastase, while having low inhibitory activity against protease 3, and surprisingly low against porcine pancreatic elastase (PPE) Has inhibitory activity. These preferred activity / selectivity profiles are due to the proper selection of a particular type of α-amino acid residue and its position in the monocyclic peptidomimetic.

  Inhibitors of proteases are cancer (RP Beckett, A. Davidson, AH Drummond, M. Whittaker, Drug Disc. Today 1996, 1, 16-26, LL Johnson, R. Dyer, DJ Hupe, Curr. Opin. Chem. Biol. 1998, 2, 466-71, D. Leung, G. Abbenante, and DP Fairlie, J. Med. Chem. 2000, 43, 305-341, T. Rockway, Expert Opin. Ther. Patents 2003, 13, 773-786), parasitic fungal and viral infections (eg schistosomiasis (MM Becker, SA Harrop, JP Dalton, BH Kalinna, DP McManus, DP Brindley, J. Biol. Chem. 1995, 270, 24496-501) , C. albicans (C. Abad-Zapetero, R. Goldman, SW Muchmore, C. Hutchins, K. Stewart, J. Navaza, CD Payne, TL Ray, Protein Sci. 1996, 5, 640-52), HIV ( A. Wlodawer, JW Erickson, Annu. Rev. Biochem. 1993, 62, 543-85, PL Darke, JR Huff, Adv. Pharmacol. 1994, 5, 399-454), hepatitis virus (JL Kim, KA Morgenstern, C Lin, T. Fox, MD Dwyer, JA Landro, SP Chambers, W. Markland, CA Lepre, ET O'Malley, S. L Harbeson, CM Rice, MA Murcko, PR Caron, JA Thomson, Cell, 1996, 87, 343-55, RA Love, HE Parge, JA Wickersham, Z. Hostomsky, N. Habuka, EW Moomaw, T. Adachi, Z Hostomska, Cell, 1996, 87, 331-342), herpes (W. Gibson, MR Hall, Drug. Des. Discov. 1997, 15, 39-47)) and inflammatory diseases, immunological diseases, respiratory organs Disease (PR Bernstein, PD Edwards, JC Williams, Prog. Med. Chem. 1994, 31, 59-120, TE Hugli, Trends Biotechnol. 1996, 14, 409-12), heart disease (MT Stubbs, WA Bode, Thromb Res. 1993, 69, 1-58, H. Fukami et al, Current Pharmaceutical Design 1998, 4, 439-453) and neurodegenerative abnormalities (R. Vassar, BD Bennett, S. Babu-Kahn, S Kahn, EA Mendiaz, Science, 1999, 286, 735-41)), angiogenesis (M. Kaatinen et al, Atherosklerosis 1996, 1231-2, 123-131) and multiple sclerosis (MZ Ibrahim et al. , J. Neuroimmunol 1996, 70, 131-138) It has appeared in the hope the therapeutic applications.

  Among proteases, serine proteases are important therapeutic targets. Serine proteases are trypsin-like (typically positively charged Lys / Arg is common), elastase-like (small P1 hydrophobic residues are present, depending on the substrate specificity, especially the type of residues present in P1 Or chymotrypsin-like (P1 has a large hydrophobic residue Phe / Tyr / Leu). X-ray crystal data of protease inhibitors are available on the PDB database (PDB: www.rcsb.org/pdb) as trypsin, α-chymotrypsin, γ-chymotrypsin, human neutrophil elastase, porcine pancreatic elastase , Thrombin, subtilidine, human cytomegalovirus protease, Achromobacter protease 1, human cathepsin G, glutamate specific protease, carboxypeptidase D, blood coagulation factor VIIa, porcine factor 1XA, mesenteric peptidase, HCV protease and thermitase It is done. Other therapeutically interesting serine proteases include tryptase, complement convertase, hepatitis C virus protein NS3 protease. Inhibitors of thrombin (eg JL Metha, LY Chen, WW Nichols, C. Mattsson, D. Gustaffson, TGP Saldeen, J. Cardiovasc. Pharmacol. 1998, 31, 345-51, C. Lila, P. Gloanec, L. Cadet, Y. Herve, J. Fournier, F. Leborgne, TJ Verbeuren, G. DeNanteuil, Synth. Comm. 1998, 28, 4419-29) and factor Xa (e.g. JP Vacca, Annu. Rep. Med. Chem. 1998, 33, 81-90) is under clinical evaluation as an antithrombotic agent and is an inhibitor of elastase (JR Williams, RC Falcone, C. Knee, RL Stein, AM Strimpler, B. Reaves, RE Giles, RD Krell) , Am. Rev. Respir. Dis. 1991, 144, 875-83) are in clinical trials for emphysema and other lung diseases, and tryptase inhibitors are currently being used for asthma (C. Seife, Science 1997, 277). 1602-3), urokinase inhibitors are in phase II clinical trials for breast cancer and chymase inhibitors for heart related diseases. Finally, cathepsin G, elastase and protease 3 are closely related to the regulation of the activity of cytokines and their receptors. Especially at the site of inflammation, the high concentration of these three neutrophil serine proteases (NSP) released from infiltrating polymorphonuclear cells is closely correlated over time with elevated levels of inflammatory cytokines, It has been shown strongly that these proteases are involved in the control of cytokine biological activity and availability (U. Bank, S. Ansorge, J. Leukoc. Biol. 2001, 69, 177-90). Therefore, highly selective elastase inhibitors have been identified in lung diseases such as chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis and ischemia-reperfusion injury, as well as glomerulonephritis, arthritis and bullous convulsions. Inflammatory diseases including non-infectious processes such as varicella (H. Ohbayashi, Epert Opin. Investig. Drugs 2002, 11, 965-980, B. Korkmaz, T. Moreau, F. Gauthier, Biochimie 2008, 90, 227) It becomes an effective target for new drug candidates.

One of the many protein serine protease inhibitors present is a sunflower trypsin inhibitor (SFTI) that exhibits similarity in both sequence and configuration to the trypsin-responsive loop of the Bowman-Birk family of serine protease inhibitors. -1), a 14-amino acid cyclic peptide derived from sunflower species (S. Luckett, R. Santiago Garcia, JJ Barker, AV Konarev, PR Shewry, AR Clarke, RL Brady, J. Mol. Biol. 1999) , 290, 525-533, Y.-Q.Long, S.-L. Lee, C.-Y.Lin, IJ Enyedy, S. Wang, P. Li, RB Dickson, PP Roller, Biorg. & Med. Chem. Lett. 2001, 11, 2515-2519). This inhibitor adopts a β-hairpin conformation when bound to the active site of bovine β-trypsin. SFTI-1 consists of β-trypsin (K _i <0.1 nM), cathepsin G (K _i about 0.15 nM), elastase (K _i about 105 μM), chymotrypsin (K _i about 7.4 μM) and thrombin (K _i about 136 mM). Was inhibited.

Compounds β- hairpin conformation cyclo ^{(-Xaa 1 -Xaa 2 -Thr 3 -Xaa} 4 -Ser 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Xaa 10 -Xaa 11 -Xaa 12 -Xaa 13 -) Is based on the β-hairpin loop of a naturally occurring peptide having a D-amino acid residue at number 12 and amino acid residues Thr and Ser conserved at numbers 3 and 5, respectively.

  Hairpin mimetic peptides attached to templates are available in literature (D. Obrecht, M. Altorfer, JA Robinson, Adv. Med. Chem. 1999, 4, 1-68, JA Robinson, Syn. Lett. 2000, 4, 429-44) Peptidomimetics immobilized on serine protease inhibition templates and their synthesis methods are described in International Patent Application (WO2003 / 054000 A1, WO2006 / 087001 A1) and A. Descours, K. Moehle, A. Renard, Although described in JA Robinson, ChemBioChem 2002, 3, 318-323, the molecules disclosed so far do not exhibit such a favorable activity / selectivity profile.

  The ability to create β-hairpin peptidomimetics using combinatorial and parallel synthesis methods has been shown (L. Jiang, K. Moehle, B. Dhanapal, D. Obrecht, JA Robinson, Helv. Chim. Acta. 2000). , 83, 3097-3112). The methods described herein enable the synthesis and screening of large amounts of hairpin mimetic libraries, structure-activity relationship studies, serine protease inhibitory activity with high potency and selectivity, especially in this specification. The discovery of new molecules with the properties of compounds suitable for new drugs with the preferred activity / selectivity profiles described is greatly facilitated.

The β-hairpin peptidomimetic of the present invention has the general formula cyclo (-Xaa ¹ -Xaa ² -Thr ³ -Xaa ⁴ -Ser ⁵ -Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Xaa ¹² −Xaa ¹³ −)
(I)
[Where:
Xaa ¹ is OctGly, Arg, hArg, Cha, Glu (phenethyl), or Dab (butanoyl);
Xaa ² is Glu, Val, Leu, Nle, Phe, hPhe, DiHPhe, Tyr, hTyr or Trp,;
Xaa ⁴ is Ala, AllylGly, Abu, or Val;
Xaa ⁶ is Ile or OctGly;
Xaa ⁷ is Pro;
Xaa ⁸ is Pro;
Xaa ⁹ is Gln or Tyr;
Xaa ¹⁰ is Lys or Asn;
Xaa ¹¹ is hLeu, Ser, hSer, hSer (Me), Thr, alloThr, Asn, Gln, hGln, Dap, Tyr, or His;
Xaa ¹² is ^D Pro, and
Xaa ¹³ is Pro, Tic, Glu, Asp, Ala, Val, or Lys;
However,
When Xaa ¹ is OctGly
Xaa ² is Glu or Nle;
Xaa ⁴ is Ala or Abu;
Xaa ⁶ is Ile or OctGly;
Xaa ¹⁰ is Lys;
Xaa ¹¹ is Ser, Thr, Asn, or Gln;
Xaa ¹³ is Pro, Tic, Ala, Val, or Lys, and / or
When Xaa ⁶ is OctGly
Xaa ¹ is OctGly, Arg, or Cha;
Xaa ² is Glu or Nle;
Xaa ⁴ is Ala or Abu;
Xaa ¹⁰ is Lys;
Xaa ¹¹ is Ser, Thr, Asn, Gln;
Xaa ¹³ is Pro, Tic, Ala, Val, or Lys;
When Xaa ¹¹ is Tyr or His,
Xaa ¹ is Arg, hArg, or Glu (phenethyl)]
And a pharmaceutically acceptable salt thereof.

According to the present invention, these β-hairpin peptidomimetics can be produced by a production process comprising the following steps: (a) a suitably functionalized solid support in the desired end product Derivatives in which the amino acid corresponding to Xaa ⁿ (n is 13, 8, 7, 6, 5 or 4) is appropriately N-protected (wherein any functional group present in the N-protected amino acid derivative is the same) Coupled with appropriate protection);
(B) removing the N-protecting group from the resulting product;
(C) The resulting product is a derivative in which the amino acid corresponding to Xaa ^n-1 in the desired final product is appropriately N-protected (wherein any functional group present in the N-protected amino acid derivative is (Appropriately protected as well) and appropriately coupled;
(D) removing the N-protecting group from the product obtained in step (c);
(E) a derivative in which the amino acid at position n-2 to 1 in the desired end product is appropriately N-protected (wherein any functional group present in the N-protected amino acid derivative is similarly suitably protected) A step substantially corresponding to steps (c) and (d),
(f) If n is not 13, a derivative in which the amino acid at position 13 to n + 1 in the desired final product is appropriately N-protected (wherein any functional group present in the N-protected amino acid derivative is the same) A step substantially corresponding to steps (c) and (d),
(G) Optionally, select one or several of the protected functional groups present in the molecule before removing the N-protecting group from the product obtained in step (e) or (f). And then appropriately replacing one or several moieties derived from acids, amino acids or amines with a reactive group liberated by conjugation, and removing the N-protecting group from the resulting product. ;
(H) separating the resulting product from the solid support;
(I) cyclizing the product separated from the solid support;
(J) removing any protecting groups present in the functional group of the chain of amino acid residues, and (k) converting the resulting product to a pharmaceutically acceptable salt, if desired, or pharmaceutical The resulting salt, which is acceptable or unacceptable, is converted into the corresponding free form compound or a different pharmaceutically acceptable salt.

A list of amino acids and amino acid residues suitable for the purpose of the present invention (abbreviations correspond to commonly used examples) is shown below.
3 character code 1 character code
Ala L-alanine A
Val L-Valine V
Leu L-Leucine L
Phe L-Phenylalanine F
His L-Histidine H
Tyr L-Tyrosine Y
Trp L-tryptophan W
Lys L-Lysine K
Arg L-Arginine R
Ser L-Serine S
Thr L-threonine T
Asp L-Aspartic acid D
Asn L-Asparagine N
Glu L-glutamic acid E
Gln L-Glutamine Q
Pro L-Proline P
AllylGly L-allylglycine
Abu L-α-Aminobutyric acid
OctGly L-octylglycine
Cha L-Cyclohexylalanine
Nle L-norleucine
hLeu L-homo-leucine
hPhe L-homophenylalanine
DiHPhe L-Dihomomo-phenylalanine
(2S) -2-Amino-5-phenylvaleric acid
hTyr L-homo-tyrosine
Dap L-2,3-Diaminopropionic acid
hArg L-homo-arginine
hSer L-homo-serine
hSer (Me) L-homo-O-methylserine
alloThr (2S, 3S) -2-amino-3-hydroxy-butyric acid
hGln L-homo-glutamine
Glu (phenethyl) (2S) -2-amino-5-phenethylamino-5-oxovaleric acid
Dab (butanoyl) (2S) -2-amino-4-butanamide-butyric acid
Tic (3S) -1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

In a particular embodiment of the invention, the β-hairpin peptidomimetic is a compound of general formula I and pharmaceutically acceptable salts thereof [wherein
Xaa ¹ is OctGly, Arg, hArg, or Glu (phenethyl);
Xaa ² is Glu, Nle, hTyr, or Val;
Xaa ⁴ is Ala or AllylGly;
Xaa ⁶ is Ile;
Xaa ⁷ is Pro;
Xaa ⁸ is Pro;
Xaa ⁹ is Gln or Tyr;
Xaa ¹⁰ is Lys;
Xaa ¹¹ is hLeu, Ser, Thr, Asn, Tyr, hGln, or His;
Xaa ¹² is ^D Pro, and
Xaa ¹³ is Pro;
However,
When Xaa ¹ is OctGly
Xaa ² is Glu or Nle;
Xaa ⁴ is Ala;
Xaa ⁶ is Ile;
Xaa ¹⁰ is Lys;
Xaa ¹¹ is Ser, Thr, or Asn
Xaa ¹³ is Pro, and
When Xaa ¹¹ is Tyr or His,
Xaa ¹ is Arg, hArg or Glu (phenethyl)].

In another specific embodiment of the invention, the β-hairpin peptidomimetic is a compound of general formula I and pharmaceutically acceptable salts thereof, wherein
Xaa ¹ is OctGly, Arg, or Glu (phenethyl);
Xaa ² is Glu, Nle, hTyr, or Val;
Xaa ⁴ is Ala;
Xaa ⁶ is Ile;
Xaa ⁷ is Pro;
Xaa ⁸ is Pro;
Xaa ⁹ is Gln or Tyr;
Xaa ¹⁰ is Lys;
Xaa ¹¹ is Ser, Thr, Asn, Tyr, or His;
Xaa ¹² is ^D Pro, and
Xaa ¹³ is Pro;
However,
When Xaa ¹ is OctGly
Xaa ² is Glu or Nle;
further
When Xaa ¹¹ is Tyr or His,
Xaa ¹ is Arg].

In another specific embodiment of the invention, the β-hairpin peptidomimetic is a compound of general formula I and pharmaceutically acceptable salts thereof, wherein
Xaa ¹ is Arg or hArg;
Xaa ² is Glu, Val, or hTyr;
Xaa ⁴ is Ala or AllylGly;
Xaa ⁶ is Ile;
Xaa ⁷ is Pro;
Xaa ⁸ is Pro;
Xaa ⁹ is Gln or Tyr;
Xaa ¹⁰ is Lys;
Xaa ¹¹ is hLeu, Ser, Thr, or hGln;
Xaa ¹² is ^D Pro, and
Xaa ¹³ is Pro].

In another specific aspect of the invention, the β-hairpin peptidomimetic is
Cyclo (-OctGly-Glu-Thr-Ala -Ser-Ile-Pro-Pro-Gln-Lys-Asn- D Pro-Pro-);
Cyclo (-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Tyr-Lys-Thr- ^D Pro-Pro-);
Cyclo (-OctGly-Glu-Thr-Ala -Ser-Ile-Pro-Pro-Gln-Lys-Ser- D Pro-Pro-);
Cyclo (-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr- ^D Pro-Pro-);
Cyclo (-Arg-hTyr-Thr-Ala -Ser-Ile-Pro-Pro-Gln-Lys-Tyr- D Pro-Pro-);
Cyclo (-Arg-Nle-Thr-Ala -Ser-Ile-Pro-Pro-Gln-Lys-Tyr- D Pro-Pro-);
Cyclo (-Arg-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Tyr-Lys-His- ^D Pro-Pro-);
Cyclo (-Glu (phenethyl) -Glu-Thr-Ala-Ser -Ile-Pro-Pro-Gln-Lys-Tyr- D Pro-Pro-);
Cyclo (-Arg-Val-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Tyr- ^D Pro-Pro-)
And a pharmaceutically acceptable salt thereof.

In another specific aspect of the invention, the β-hairpin peptidomimetic is
Cyclo (-Arg-Glu-Thr-AllylGly-Ser-Ile-Pro-Pro-Gln-Lys-Thr- ^D Pro-Pro-);
Cyclo (-Arg-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Tyr-Lys-Ser- ^D Pro-Pro-);
Cyclo (-hArg-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr- ^D Pro-Pro-);
Cyclo (-Arg-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-hLeu- ^D Pro-Pro-);
Cyclo (-Arg-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr- ^D Pro-Pro-);
Cyclo (-Arg-Glu-Thr-Ala -Ser-Ile-Pro-Pro-Tyr-Lys-hGln- D Pro-Pro-);
Cyclo (-Arg-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Tyr-Lys-Thr- ^D Pro-Pro-);
Cyclo (-Arg-Val-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr- ^D Pro-Pro-);
Cyclo (-Arg-hTyr-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr- ^D Pro-Pro-)
And a pharmaceutically acceptable salt thereof.

  The production method of the present invention can be conveniently carried out as a parallel array synthesis to obtain a library of β-hairpin peptidomimetics of general formula I above. By such parallel synthesis, an array of many compounds of general formula I (usually 24 to 192, typically 96) can be obtained in high yields and defined purities, dimers and polymers. It is possible to minimize the formation of by-products. Proper selection of the solid support (ie, solid support + linker molecule), template and cyclization site thereby plays an important role.

The functionalized solid support is conveniently derived from polystyrene cross-linked with divinylbenzene (preferably 1-5%), polystyrene coated with a polyethylene glycol spacer (Tentagel®), and polyacrylamide resin. (D. Obrecht, JM Villalgordo, “Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries”, Tetrahedron Organic Chemistry Series, Vol. 17, Pergamon, Elsevier Science, 1998).
The solid support is a bifunctional (i.e., an anchor group for attachment to the solid support at one end and a selectively cleavable functional group at the other end for subsequent chemical transformations and cleavage procedures. Functionalized with spacer molecules). In the present invention, two types of linkers are used:

Type 1 linkers are designed to release amide groups under acidic conditions (H. Rink, Tetrahedron Lett. 1987, 28, 3783-3790). This type of linker forms an amide of the carboxyl group of an amino acid. Examples of resins functionalized with such a linker structure include:
4-[((((2,4-Dimethoxy-phenyl) Fmoc-aminomethyl) phenoxyacetamido) aminomethyl] PS resin, 4-[((((2,4-dimethoxyphenyl) Fmoc-aminomethyl) phenoxy-acetamido) Aminomethyl] -4-methyl-benzhydrylamine PS resin (Rink amide MBHA PS resin), and 4-[(((2,4-dimethoxy-phenyl) Fmoc-aminomethyl) phenoxyacetamido) aminomethyl] benzhydrylamine PS resin (Rink amide BHA PS resin) may be mentioned. Preferably, the support is cross-linked with divinylbenzene (most preferably 1-5%) and functionalized with a 4-(((2,4-dimethoxyphenyl) Fmoc-aminomethyl) phenoxyacetamide) linker. Derived from polystyrene.

  Type 2 linkers are designed to ultimately release carboxyl groups under acidic conditions. This type of linker forms an acid labile ester (generally acid labile benzyl, benzhydryl and trityl esters) with amino acid carboxyl groups, such as 2-methoxy-4 -Hydroxymethylphenoxy (Sasrin® linker), 4- (2,4-dimethoxyphenyl-hydroxymethyl) -phenoxy (Rink linker), 4- (4-hydroxymethyl-3-methoxyphenoxy) butyric acid (HMPB linker) ), Trityl and 2-chlorotrityl. Preferably, the support is derived from polystyrene cross-linked with divinylbenzene (most preferably 1-5%) and functionalized with a 2-chlorotrityl linker.

  When carried out as a parallel synthesis, the process of the invention can be conveniently carried out as described herein below, but if it is desired to synthesize a single compound of the invention, these methods It will be readily apparent to those skilled in the art how the procedure must be modified.

  A number of reaction vessels equal to the total number of compounds synthesized by the parallel method are loaded with 25-1000 mg, preferably 60 mg, of a suitable functionalized solid support, preferably 1-5% cross-linked polystyrene or Tentajel resin. .

  The solvent used must be capable of swelling the resin, dichloromethane (DCM), dimethylformamide (DMF), n-methylpyrrolidone (NMP), dioxane, toluene, tetrahydrofuran (THF), ethanol (EtOH ), Trifluoroethanol (TFE), isopropyl alcohol, and the like, but are not limited thereto. Solvent mixtures containing a polar solvent (eg, 20% TFE / DCM, 35% THF / NMP) as at least one component are beneficial in ensuring high reactivity and solvation of the peptide chains attached to the resin ( GB Fields, CG Fields, J. Am. Chem. Soc. 1991, 113, 4202-4207). The development of various linkers that release the C-terminal carboxylic acid group under mild acidic conditions that protect the side-chain functional groups and do not affect acid-labile groups There is considerable progress in synthesis. A linker derived from 2-methoxy-4-hydroxybenzyl alcohol (Sasrin® linker, Mergler et al., Tetrahedron Lett. 1988, 29 4005-4008) is diluted with dilute trifluoroacetic acid (0.5-1% in DCM). It is cleavable with TFA) and is stable to Fmoc elimination conditions during peptide synthesis, and further protecting groups based on Boc / tBu are compatible with this protection scheme. Other linkers suitable for the production method of the present invention include a super strong acid labile 4- (2,4-dimethoxyphenyl-hydroxymethyl) -phenoxy linker (Rink linker, H. Rink, Tetrahedron Lett. 1987, 28 , 3787-3790) (10% acetic acid in DCM or 0.2% trifluoroacetic acid in DCM required for peptide elimination), linker (HMPB) derived from 4- (4-hydroxymethyl-3-methoxyphenoxy) butyric acid -Linker, Florsheimer & Riniker, Peptides 1991, 1990 131) (cleaved with 1% TFA / DCM to obtain peptide fragments containing all acid labile side chain protecting groups), and further 2-chlorotrityl chloride Linker (Barlos et al., Tetrahedron Lett. 1989, 30, 3943-3946) (Peptide can be removed using a mixture of glacial acetic acid / trifluoroethanol / DCM (1: 2: 7) for 30 minutes) It is done.

Suitable protecting groups for amino acids and their respective residues are, for example:
-Protecting groups for amino groups (for example also present in the side chain of lysine)
Cbz benzyloxycarbonyl
Boc tert-butyloxycarbonyl
Fmoc 9-Fluorenylmethoxycarbonyl
Alloc aroylcarbonyl
Teoc trimethylsilylethoxycarbonyl
Tcc Trichloroethoxycarbonyl
Nps o-nitrophenylsulfonyl;
Trt triphenylmethyl or trityl;

A protecting group (by conversion to an ester with an alcohol component) of a carboxyl group (eg also present in the side chain of aspartic acid and glutamic acid)
tBu tert-butyl
Bn benzyl
Me methyl
Ph phenyl
Pac Phenacyl
Allyl
Tse Trimethylsilylethyl
Tce trichloroethyl;

A protecting group for a guanidino group (for example present in the side chain of arginine)
Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonyl
Ts tosyl (ie p-toluenesulfonyl)
Cbz benzyloxycarbonyl
Pbf pentamethyldihydrobenzofuran-5-sulfonyl;

-Protecting groups for hydroxy groups (eg present in the side chain of threonine and serine)
tBu tert-butyl
Bn benzyl
Trt Trityl.

9-Fluorenylmethoxycarbonyl- (Fmoc) -protected amino acid derivatives are preferably used as building blocks in the construction of the β-hairpin loop mimics of the present invention. Deprotection, i.e. cleavage of the Fmoc group, the, 2% DBU / 2% piperidine 20% piperidine or in DMF in DMF, and 25% hexafluoroisopropanol in CH ₂ Cl ₂ to be used.

  The amount of reactant, i.e., amino acid derivative, is 1 to 20 equivalents based on milliequivalents / gram (meq / g) of the functionalized solid support initially weighed into the reaction tube (typically 0.1-2.85 meq / g polystyrene resin). If desired, an additional equivalent of reactants can be used to operate the reaction in a reasonable time. A preferred workstation (but not limited to) is the Labsource's Combi-chem station, Protein Technologies' Symphony and MultiSyn Tech's-Syro synthesizer, which is a straight line that is fully protected from the solid support. During the process of desorbing the chain peptide, a transfer unit and a reservoir box are further provided. All synthesizers can provide a controlled environment, for example, the reaction can be carried out at a temperature different from room temperature and optionally under an inert gas atmosphere.

  Formation of the amide bond requires activation of the α-carboxyl group for the acylation step. This activation can be achieved by dicyclohexylcarbodiimide (DCC, Sheehan & Hess, J. Am. Chem. Soc. 1955, 77, 1067-1068) or diisopropylcarbodiimide (DIC, Sarantakis et al Biochem. Biophys. Res. Commun. 1976, 73 , 336-342), and the like, the obtained dicyclohexylurea and diisopropylurea are insoluble and soluble in commonly used solvents, respectively. An alternative to the carbodiimide method includes 1-hydroxybenzotriazole (HOBt, Koenig & Geiger, Chem. Ber. 1970, 103, 788-798) as an additive to the coupling mixture. HOBt acts as a catalyst to prevent dehydration, suppress racemization of activated amino acids, and improve slow coupling reactions. Certain phosphonium reagents can be combined with direct coupling agents such as benzotriazole-1-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate (BOP, Castro et al., Tetrahedron Lett. 1975, 14, 1219-1222). , Synthesis 1976, 751-752), or benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (Py BOP, Coste et al., Tetrahedron Lett. 1990, 31, 205-208), or 2 -(1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) or hexafluorophosphate (HBTU, Knorr et al., Tetrahedron Lett. 1989, 30, 1927 -1930), these phosphonium reagents are also suitable for in situ formation of HOBt esters with protected amino acid derivatives. More recently, diphenoxyphosphoryl azide (DPPA) or O- (7-azabenzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium tetrafluoroborate (TATU), or O— (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (HATU) / 7-aza-1-hydroxybenzotriazole (HOAt, Carpino et al., Tetrahedron Lett. 1994, 35, 2279 2281) or (6-Chloro-1H-benzotriazol-1-yl-)-N, N, N ', N'-1,1,3,3-tetramethyl-uronium Tetrafluoroborate (TCTU) or hexafluorophosphate (HCTU, Marder, Shivo and Albericio: HCTU and TCTU: New Coupling Reagents: Development and Industrial Applications, Poster Presentation, Gordon Conference February 2002) are also used as coupling agents. , 1,3,3-Bis (tetramethylene) chlorouronium hexa Luo-phosphate (PyClU) (especially for coupling of N-methylated amino acids) (J. Coste, E. Frerot, P. Jouin, B. Castro, Tetrahedron Lett. 1991, 32, 1967) or pentafluorophenyl diphenyl -Phosphinates (S. Chen, J. Xu, Tetrahedron Lett. 1991, 32, 6711) are also used.

  From the fact that a nearly quantitative coupling reaction is essential, it is desirable to obtain experimental evidence that the reaction is complete. A ninhydrin test (Kaiser et al., Anal. Biochemistry 1970, 34, 595), where a positive colorimetric reaction to an aliquot of peptide bound to the resin quantitatively indicates the presence of primary amines, is easy after each coupling step. Can be implemented promptly. Fmoc chemistry allows spectrophotometric detection of the Fmoc chromophore when released with a base (Meienhofer et al., Int. J. Peptide Protein Res. 1979, 13, 35-42).

By repeatedly exposing to pure solvent in one of the two ways described below, the intermediate bound to the resin in each reaction vessel is washed to remove excess residual reagents, solvents, and by-products.
1) Fill the reaction vessel with solvent (preferably 5 mL) and stir for 5 to 300 minutes, preferably 15 minutes, then drain to remove the solvent.
2) Fill the reaction vessel with solvent (preferably 5 mL) and drain into a receiving vessel such as a test tube or vial.
Both washing procedures described above are repeated up to about 50 times (preferably about 10 times), and the removal efficiency of reagents, solvents and by-products is monitored by methods such as TLC, GC, or inspection of the wash.

  The above procedure, which reacts the compound bound to the resin in the reaction well with the reagent and then removes excess reagent, by-products, and solvent, results in the fully protected linear peptide finally bound to the resin. Repeat the series of transformations until it is obtained.

Prior to detachment of this fully protected linear peptide from the solid support, one or several protected functional groups present in the molecule are optionally deprotected and deprotected, if desired. The reactive group can be appropriately substituted. In order to do this, it is first necessary to replace the functional group of interest with a protecting group that can be selectively removed without affecting the other protecting groups present.
Alloc (aroylcarbonyl) is an example of an amino protecting group and allyl is an example of a carboxylic acid protecting group. Either group can be selectively removed using Pd and phenylsilane in CH ₂ Cl ₂ without affecting other protecting groups present in the molecule (eg, Fmoc). The reactive group thus deprotected can then be treated with a reagent suitable for introducing the desired substituent. That is, for example, an amino group can be acylated using an acylating agent corresponding to the acyl substituent to be introduced, and a carboxyl group can be derivatized by introducing an amino substituent. Preferably, Alloc or allyl is eliminated by applying 0.2 equivalents of tetrakis (triphenyl-phosphine) palladium (0) (10 mM) and 10 equivalents of phenylsilane in dry CH ₂ Cl ₂ at room temperature for 15 minutes. To do. After filtration and resin washing, deprotection is performed by repeating this procedure with fresh reagent solution. In the case of a deprotected carboxy group, for example, subsequent amine coupling can be performed, for example, by applying the reagents / reaction conditions for amide bond formation described above.

  Desorption of the fully protected linear peptide from the solid support is accomplished by exposing the loaded resin to a solution of the reagent used for cleavage (preferably 3-5 mL). Temperature control, stirring, and reaction monitoring are performed as described above. The reaction vessel is connected to a reservoir box including a reservoir tube via a transfer unit, and the cleaved product solution is efficiently recovered. Next, the resin remaining in the reaction vessel is washed 2 to 5 times with 3 to 5 mL of an appropriate solvent as described above, and the separated product is extracted (washed out) as much as possible. The resulting product solution is mixed with care to avoid contamination. The individual compounds / extracts are then manipulated as necessary to isolate the final compound. Typical operations include, but are not limited to, evaporation, concentration, liquid / liquid extraction, acidification, basification, neutralization, or further reaction in solution.

  The solution containing the fully protected linear peptide derivative cleaved from the solid support and neutralized with base is evaporated. Next, cyclization is carried out in solution using solvents such as DCM, DMF, dioxane, THF. The various coupling agents described above can be used for cyclization. The duration of cyclization is about 6 to 48 hours, preferably about 16 hours. The reaction is allowed to proceed, followed by, for example, RP-HPLC (reverse phase high performance liquid chromatography). The solvent is then removed by evaporation and the fully protected cyclic peptide derivative is dissolved in a water immiscible solvent (eg DCM) and the solution is extracted with water or a mixture of water miscible solvents and excess Remove the coupling agent.

Finally, the fully protected peptide derivative is treated with 95% TFA, 2.5% H ₂ O, 2.5% TIS, or another scavenger mixture to effect cleavage of the protecting group. The cleavage reaction time is usually 30 minutes to 12 hours, preferably about 2.5 hours.
Alternatively, complete deprotection of the peptide from the solid support and complete deprotection can be accomplished manually in a glass container.

After complete deprotection, for example, the following method can be used for further workup:
1) Evaporate the volatiles to dryness and dissolve the crude peptide in 20% AcOH in water and extract with isopropyl ether or other suitable solvent. The aqueous layer was collected and evaporated to dryness, and the fully deprotected peptide, cyclo (-Xaa ¹ -Xaa ² -Thr ³ -Xaa ⁴ -Ser ⁵ -Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ^{10 -Xaa 11 -Xaa 12 -Xaa 13 -} ) to obtain as a final product;
2) Concentrate the deprotection mixture under reduced pressure. After precipitation of the fully deprotected peptide, preferably in diethyl ether at 0 ° C., the solid is washed up to about 10 times, preferably 3 times, and dried to give a fully deprotected peptide, cyclo ^{(-Xaa 1 -Xaa 2 -Thr 3 -Xaa} 4 -Ser 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Xaa 10 -Xaa 11 -Xaa 12 -Xaa 13 -) to obtain as a final product.

Depending on the purity, the final product obtained as described above can be used directly in biological assays or needs to be further purified (eg by preparative HPLC).
As described above, optionally, the resulting fully deprotected cyclic product is then converted to a pharmaceutically acceptable salt, or the resulting pharmaceutically acceptable or unacceptable salt. Can be converted to the corresponding free compound or a different pharmaceutically acceptable salt. Any of these operations can be performed by methods well known in the art.

  The β-hairpin peptidomimetic of the present invention is an inflammatory disease or lung disease or infection or immunological disease or heart disease or neurodegenerative disease mediated by or caused by serine protease activity, or cancer is serine It can be used in a wide range of applications mediated by or due to protease activity. For the control or prevention of diseases or disorders suitable for treatment with protease inhibitors, the β-hairpin peptidomimetics of the invention can be administered per se or can be carriers, diluents or It can be applied as an appropriate formulation together with excipients.

  α1 antitrypsin deficiency (AATD), emphysema, rheumatoid arthritis, osteoarthritis, atherosclerosis, psoriasis, cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), bronchiectasis, bronchodilation, chronic bronchitis, multiple sclerosis, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), pulmonary hypertension (PH), pulmonary arterial hypertension (PAH), Pancreatitis, asthma, allergic rhinitis, inflammatory skin disease, restenosis after angioplasty, systemic inflammatory response syndrome (SIRS), ischemia-reperfusion injury, cardiac hypertrophy, myocarditis, acute myocardial infarction (AMI), Heart failure, heart transplantation, inflammatory bowel disease (IBD), colitis, Crohn's disease, adaptive colitis or cancer (including but not limited to lung cancer, breast cancer or cancer associated with angiogenesis or metastasis), etc. Treatment, prevention, regulation of various diseases , Or when used in remodeling, the β-hairpin peptidomimetics of the present invention alone, as a mixture of several β-hairpin peptidomimetics, in combination with other anti-inflammatory or antibacterial or anticancer agents, and / or It can be administered in combination with other pharmaceutically active agents. The β-hairpin peptidomimetics of the present invention can be administered per se or as a pharmaceutical composition.

  Pharmaceutical compositions containing the β-hairpin peptidomimetics of the present invention are manufactured by conventional mixing, dissolving, granulating, coated tablet making, wet grinding, emulsifying, encapsulating, encapsulating, or lyophilizing processes. be able to. The pharmaceutical composition comprises one or more physiologically acceptable carriers, diluents, excipients that facilitate the processing of the active β-hairpin peptidomimetic into a pharmaceutically usable preparation. It can be formulated by a conventional method using a form or an auxiliary agent. Proper formulation is dependent upon the method of administration chosen.

  The β-hairpin peptidomimetics of the present invention for topical administration can be formulated as solutions, gels, ointments, creams, suspensions, etc. well known in the art.

  Systemic formulations include those designed for administration by injection, eg, subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection, and designed for transdermal, transmucosal, oral, or pulmonary administration The thing which was done is mentioned.

The β-hairpin peptidomimetics of the present invention for injection can be formulated in a suitable solution, preferably a physiologically compatible buffer such as Hanks's solution, Ringer's solution, or physiological saline buffer. . The solution can contain pharmaceutical reagents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the β-hairpin peptidomimetics of the present invention may be in the form of a powder for mixing with a suitable vehicle, such as sterile pyrogen-free water, prior to use.
For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation, as is known in the art.

  For oral administration, the compounds can be readily formulated by mixing an active β-hairpin peptidomimetic of the present invention with pharmaceutically acceptable carriers known in the art. Such carriers include tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions for ingestion of the β-hairpin peptidomimetics of the present invention by the patient being treated. It makes it possible to formulate as an agent, powder, etc. Suitable excipients for oral formulations such as powders, capsules and tablets include fillers such as sugars (such as lactose, sucrose, mannitol, and sorbitol), cellulose preparations (corn starch, wheat starch, Rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP)), granulating agents, and binders. If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, the solid dosage form can be sugar coated or enteric coated using standard techniques.

  Suitable carriers, excipients, or diluents for oral liquid preparations such as suspensions, elixirs and solutions include water, glycols, oils, alcohols and the like. Furthermore, flavoring agents, preservatives, coloring agents and the like can be added.

  Compositions for buccal administration can take the form of tablets, lozenges and the like formulated as is known in the art.

  For administration by inhalation, a suitable propellant (eg, dichlorodifluoromethane, trichlorofluoromethane, carbon dioxide or other suitable gas) is used to form the β- of the present invention in the form of an aerosol spray from a pressurized pack or nebulizer. Hairpin peptidomimetics are conveniently delivered. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. A capsule or cartridge, such as gelatin, for use in an inhaler or insufflator, containing a powder mixture of the β-hairpin peptidomimetic of the invention and a suitable powder base (such as lactose or starch) may also be formulated. Good.

  The compounds can also be formulated in rectal or vaginal compositions such as suppositories, with appropriate suppository bases such as cocoa butter or other glycerides.

  In addition to the above formulations, the β-hairpin peptidomimetics of the present invention can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) and by intramuscular injection. In the manufacture of such depot formulations, the β-hairpin peptidomimetics of the present invention can be prepared using suitable polymers or hydrophobic substances (eg as acceptable emulsions in oil) or ion exchange resins, or sparingly soluble salts. Can be formulated as

  In addition, other pharmaceutical delivery systems such as liposomes and emulsions well known in the art can be used. Certain organic solvents (eg, dimethyl sulfoxide) can also be used. Furthermore, the β-hairpin peptidomimetics of the present invention can be delivered using a sustained release system (eg, a solid polymer semi-permeable matrix containing a therapeutic agent. Various sustained release materials have been established and will be known to those skilled in the art. Sustained release capsules can release compounds for weeks to 100 days or more depending on their chemical properties, depending on the chemical nature and biological stability of the therapeutic agent, Additional strategies for protein stabilization can be used.

  Since the β-hairpin peptidomimetics of the present invention contain charged residues, they can be included in any of the above formulations as such or as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts are more soluble in aqueous or other protic solvents than in the corresponding free form. Particularly suitable pharmaceutically acceptable salts include carboxylic acids, phosphonic acids, sulfonic acids and sulfamic acids such as acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid. , Adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, amino acids (such as glutamic acid or aspartic acid), maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid Acid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methane- or ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 2- Naphthalenesulfonic acid, 1,5-naphtha Disulfonic acid, 2-, 3- or 4-methyl-benzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamic acid, and Examples thereof include salts with other organic protonic acids (such as ascorbic acid). Suitable inorganic acids are, for example, hydrohalic acids (such as hydrochloric acid), sulfuric acid and phosphoric acid.

  The β-hairpin peptidomimetics of the present invention, compositions thereof, are generally used in an amount effective to achieve the intended purpose. It will be appreciated that the amount used will depend on the specific application.

  A therapeutically effective amount for topical administration for treatment or prevention of a disease suitable for treatment with a beta hairpin mimetic can be determined using, for example, the in vitro assays described in the Examples. Treatment can be applied while the disease is manifested or even when it is not manifested. One of ordinary skill in the art will be able to determine a therapeutically effective amount for locally treating the disease without undue experimentation.

  A therapeutically effective dose for topical administration for treatment or prevention can be determined, for example, using the in vitro assays described in the Examples. Treatment can be applied during or when HIV infection is manifested. One skilled in the art will be able to determine a therapeutically effective amount for treating local HIV infection without undue experimentation.

A therapeutically effective dose for systemic administration can be estimated initially from in vitro assays. For example, a dose can be formulated in an animal model to achieve a concentration range of circulating β-hairpin peptidomimetic that includes an IC ₅₀ determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
Initial doses can also be determined from in vivo data (eg, animal models) using techniques well known in the art. One skilled in the art could readily optimize administration to humans based on animal data.
The applied dose as a serine protease can be individually adjusted to provide a plasma concentration of the β-hairpin peptidomimetic of the present invention sufficient to maintain a therapeutic effect. The therapeutically effective serum concentration can be achieved by administering multiple doses daily.
In cases of local administration or selective uptake, the effective local concentration of the β-hairpin peptidomimetics of the present invention may not be related to plasma concentration. One skilled in the art will be able to optimize therapeutically effective local concentrations without undue experimentation.
Of course, the amount of β-hairpin peptidomimetic administered will depend on the subject being treated, the subject's weight, the severity of the illness, the method of administration, and the judgment of the prescribing physician.

  In general, a therapeutically effective amount of a β-hairpin peptidomimetic described herein will provide a therapeutic effect without causing substantial toxicity.

The toxicity of the β-hairpin peptidomimetics of the present invention uses cell cultures or experimental animals such as determining LD ₅₀ (lethal dose for 50% of population) or LD ₁₀₀ (lethal dose for 50% of population). It can be determined by standard pharmaceutical procedures. The dose ratio between toxic and therapeutic effects is the therapeutic index. Compounds that exhibit high therapeutic indices are preferred. Data obtained from these cell culture assays and animal studies can be used to formulate a non-toxic dose range for humans. The dose of the β-hairpin peptidomimetic of the present invention is preferably within a range of circulating concentrations, including an effective dose with little or no toxicity. The dosage may vary within the range depending on the dosage form used and the administration route used. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, eg, Fingl et al. 1975, The Pharmacological Basis of Therapeutic, Ch. 1, p. 1) ).

The present invention is enriched with atoms having mass numbers or masses different from those normally found in nature (eg ² H (D), ³ H, ¹¹ C, ¹⁴ C, ¹²⁹ I, etc.). compounds) except the general formula: cyclo ^{(-Xaa 1 -Xaa 2 -Thr 3 -Xaa} 4 -Ser 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Xaa 10 -Xaa 11 -Xaa 12 -Xaa ^The same compound as the compound shown by 13-) is included. These isotope analogs and pharmaceutical salts and formulations thereof are not limited thereto, but may be therapeutic and / or diagnostic if, for example, an in vivo half-life fine-tuning results in an optimized dosing schedule It is considered to be a useful substance.
The following examples illustrate the invention, but are not intended to limit the scope of the invention in any way.

1. Peptide synthesis Coupling of initially protected amino acid residues to resin 1 g (1.4 mMol) 2-chlorotrityl chloride resin (1.4 mMol / g, 100-200 mesh, copoly (styrene-1% DVB) polymer Matrix, Barlos et al. Tetrahedron Lett. 1989, 30, 3943-3946) was placed in a dry flask. The resin was suspended in CH ₂ Cl ₂ (5 mL) and allowed to swell for 30 minutes at room temperature under continuous shaking. A solution of 0.98 mMol (0.7 equiv) initially suitably protected amino acid residue (see below) in CH ₂ Cl ₂ (5 mL) mixed with 960 μl (4 equiv) diisopropylethylamine (DIEA). added. After the reaction mixture was shaken at 25 ° C. for 4 hours, the resin was filtered and washed sequentially with CH ₂ Cl ₂ (1 ×), DMF (1 ×), and CH ₂ Cl ₂ (1 ×). A solution of CH ₂ Cl ₂ / MeOH / DIEA (17/2/1, 10 mL) was added to the resin and the suspension was shaken for 30 minutes. After filtration, the resin was CH ₂ Cl ₂ (1x), DMF (1x), CH ₂ Cl ₂ (1x), MeOH (1x), CH ₂ Cl ₂ (1x), MeOH (1x), CH ₂ Cl ₂ (2x ), Et ₂ O (2 ×), and then dried under vacuum for 6 hours.
A typical filling amount was 0.6 to 0.7 mMol / g.

The following pre-filled resins were prepared:
Fmoc-Ser (tBu) -O-2-chlorotrityl resin and Fmoc-Pro-O-2-chlorotrityl resin.
Synthesis was performed using a Syro peptide synthesizer (MultiSynTech) using a 24-96 reaction vessel. In each container, 0.04 mMol of the above resin was placed, and the resin was swollen with CH ₂ Cl ₂ and DMF for 15 minutes, respectively. The following reaction cycle was programmed and carried out.
Step Reagent Time 1 DMF, Wash 5x1 min 2 20% Piperidine / DMF 1x5 min, 1x15 min 3 DMF, Wash 5x1 min 4 3.6 eq Fmoc amino acid, 3.6 eq HOAT / DMF
+3.6 equivalents DIC / DMF 1x40 min 5 DMF, wash 1x1 min 6 3.6 equivalents Fmoc amino acid, 3.6 equivalents HOAT / DMF
+3.6 equivalent HATU
+7.2 equivalents DIPEA 1x40 min

Unless otherwise noted, Fmoc deprotection was performed by applying steps 1-3 of the above reaction cycle following the final coupling of amino acids.
Appropriately protected amino acid building blocks are commercially available or can be synthesized as is known in the art.

Procedure A for coupling phenethylamine to a side chain having a carboxyl group
Binding of phenethylamine to linear peptides selectively deprotected on the resin:
In order to remove the allyl protecting group from the carboxy functionality present in the peptide bound to the resin, the resin (0.04 mMol) was swollen with freshly distilled CH ₂ Cl _{2 for} at least 15 minutes and then dried CH _2. 0.2 equivalents of tetrakis (triphenyl-phosphine) palladium (0) (10 mM) and 10 equivalents of phenylsilane in Cl ₂ were added. After the reaction mixture was shaken for 15 minutes at room temperature, the resin was filtered and the procedure was repeated with fresh addition of reagent solution. After the resin was washed sequentially with CH ₂ Cl ₂ , DMF and Et ₂ O, the resin was swollen again with CH ₂ Cl ₂ followed by 3.6 equivalents of phenethylamine and 3.6 equivalents dissolved in DMF. A mixture of HOAt and 3.6 equivalents of DIC dissolved in DMF was added, and the reaction mixture was allowed to stand for 1 hour except for occasional stirring to bind phenethylamine. After filtration and washing the resin three times with DMF, the procedure is repeated using a mixture of 3.6 equivalents of the same amine and 3.6 equivalents HOAt dissolved in DMF and a mixture of 3.6 equivalents HATU and 7.2 equivalents DIPEA dissolved in DMF. This completes the coupling.

Post-treatment of the cyclized and cyclized peptide backbone Cleavage of the fully protected peptide fragment After the synthesis was complete, the resin (0.04 mMol) was washed with 1% TFA, 1 mL in CH ₂ Cl ₂ (v / v) ( Suspended in 0.13 mMol, 3.4 eq) for 3 min, filtered, and then the filtrate was neutralized with 1 mL (0.58 mMol, 14.6 eq) of 10% DIEA in CH ₂ Cl ₂ (v / v) did. This procedure was repeated three times to ensure complete cleavage. The filtrate is evaporated to dryness and a sample of the product is completely deprotected using a cleavage mixture containing 95% trifluoroacetic acid (TFA), 2.5% water, and 2.5% triisopropylsilane (TIS). The efficiency of linear peptide synthesis was monitored by analysis by reverse phase HPLC ( _C18 column) and ESI MS.

Cyclization of linear peptide Fully protected linear peptide (0.04 mMol) was dissolved in DMF (4 μMol / mL). Then 30.4 mg (0.08 mMol, 2 eq) HATU, 10.9 mg (0.08 mMol, 2 eq) HOAt and 28 μl (0.16 mMol, 4 eq) DIEA were added and the mixture was added at 25 ° C. Vortexed for 16 hours and then concentrated under high vacuum. The residue was partitioned between CH ₂ Cl ₂ and H ₂ O / CH ₃ CN (90/10: v / v). The CH ₂ Cl ₂ phase was evaporated to give a fully protected cyclic peptide.

Complete deprotection of the cyclic peptide Cleavage of the resulting cyclic peptide containing 82.5% trifluoroacetic acid (TFA), 5% water, 5% thioanisole, 5% phenol, and 2.5% ethanedithiol (EDT) The mixture was dissolved in 3 mL. The mixture was left at 25 ° C. for 2.5 hours and then concentrated under vacuum. After the fully deprotected cyclic peptide was precipitated with diethyl ether (Et ₂ O) at 0 ° C., the solid was washed twice with Et ₂ O and then dried.
After the composition was purified by preparative HPLC, the peptide was lyophilized (white powder) and analyzed by the following analytical method.

Analysis method A (Examples 5 to 10, 16 to 18)
Analytical HPLC retention time (RT, min) was measured using an Ascentis Express C18 column, 50 x 3.0 mm, (cod. 53811-U-Supelco), solvent A (H ₂ O + 0.1% TFA) and B (CH ₃ CN + 0.01% TFA) was used with the following gradient: 0-0.05 min: 97% A, 3% B, 4.95 min: 3% A, 97% B, 5. 35 minutes: 3% A, 97% B, 5.40 minutes: 97% A, 3% B.
Flow rate = 1.3 mL / min, UV_Vis = 220 nm.

Analysis method B (Examples 1 to 4)
Analytical HPLC retention time (RT, min) was measured using an Ascentis Express C18 column, 50 x 3.0 mm, (cod. 53811-U-Supelco), solvent A (H ₂ O + 0.1% TFA) and B (CH ₃ CN + 0.01% TFA) was used with the following gradient: 0-0.05 min: 97% A, 3% B, 3.40 min: 33% A, 67% B, 45 minutes: 3% A, 97% B, 3.65 minutes: 3% A, 97% B, 3.70 minutes: 97% A, 3% B.
Flow rate = 1.3 mL / min, UV_Vis = 220 nm.

Analysis method C (Examples 11 to 15)
Analytical HPLC retention time (RT, min) was measured using an Xselect CSH C18 XP column, 100 × 3.0 mm, (cod. 186006107, Waters), with solvent A (H ₂ O + 0.1% TFA) and B ( CH ₃ CN + 0.01% TFA) was used with the following gradient: 0-0.05 min: 95% A, 5% B, 10.05 min: 3% A, 97% B, 12.05 Minutes: 3% A, 97% B, 12.10 minutes: 95% A, 5% B.
Flow rate = 0.6 mL / min, UV_Vis = 220 nm.

Examples 1 to 13 are shown in Table 1. The peptide was synthesized as follows:
The starting resin was Fmoc-Ser (tBu) -O-2-chlorotrityl prepared as described above. Xaa ⁴ (which finally becomes 4-position) was bonded to this resin. A linear peptide of the following sequence was synthesized on a solid support according to the above procedure: Resin-Ser ⁵ -Xaa ⁴ -Thr ³ -Xaa ² -Xaa ¹ -Xaa ¹³ -Xaa ¹² -Xaa ¹¹ -Xaa ¹⁰ -Xaa ⁹ -Xaa ⁸ -Xaa ⁷ -Xaa ⁶ . After final Fmoc deprotection as described above, the peptide was cleaved from the resin as described above and cyclized, deprotected and purified.
Table 1 shows the HPLC retention time and UV-purity measured using the analytical method described above.

Examples 14 and 16 are shown in Table 1. The peptide was synthesized as follows:
The starting resin was Fmoc-Pro-O-2-chlorotrityl resin prepared as described above. Xaa ¹² (finally at position 12) was bound to this resin. A linear peptide of the following sequence was synthesized on a solid support according to the above procedure: Resin-Pro ¹³ -Xaa ¹² -Xaa ¹¹ -Xaa ¹⁰ -Xaa ⁹ -Xaa ⁸ -Xaa ⁷ -Xaa ⁶ -Ser ⁵ -Xaa ⁴ -Thr ³ -Xaa ² -Xaa ¹ . After final Fmoc deprotection as described above, the peptide was cleaved from the resin as described above and cyclized, deprotected and purified.
Table 1 shows the HPLC retention time and UV-purity measured using the analytical method described above.

Example 15 is shown in Table 1. The peptide was synthesized as follows:
The starting resin was Fmoc-Pro-O-2-chlorotrityl resin prepared as described above. Xaa ¹² (finally at position 12) was bound to this resin. A linear peptide of the following sequence was synthesized on a solid support according to the above procedure: Resin-Pro ¹³ -Xaa ¹² -Xaa ¹¹ -Xaa ¹⁰ -Xaa ⁹ -Xaa ⁸ -Xaa ⁷ -Xaa ⁶ -Ser ⁵ -Xaa ⁴ -Ser ³ -Xaa ² -Glu ¹ . Prior to final Fmoc deprotection, procedure A was applied to couple phenylethylamine to the side chain of Glu ¹ . After the final Fmoc deprotection, the peptide was cleaved from the resin as described above and cyclized, deprotected and purified.
Table 1 shows the HPLC retention time and UV-purity measured using the analytical method described above.

Examples 17-18 are shown in Table 1. The peptide was synthesized as follows:
The starting resin was Fmoc-Pro-O-2-chlorotrityl resin prepared as described above. Xaa ⁷ (which finally becomes 7-position) was bound to this resin. A linear peptide of the following sequence was synthesized on a solid support according to the above procedure: Resin-Pro ⁸ -Xaa ⁷ -Xaa ⁶ -Ser ⁵ -Xaa ⁴ -Thr ³ -Xaa ² -Xaa ¹ -Xaa ¹³ -Xaa ¹² -Xaa ¹¹ -Xaa ¹⁰ -Xaa ⁹ . After final Fmoc deprotection as described above, the peptide was cleaved from the resin as described above and cyclized, deprotected and purified.
Table 1 shows the HPLC retention time and UV-purity measured using the analytical method described above.

2. Biological methods 2.1. Preparation of peptide samples Lyophilized peptides were weighed with Microbalance (Mettler MT5) and dissolved in DMSO to a final concentration of 10 mM. The stock solution was stored at + 4 ° C. protected from light. Biological analysis was performed under analytical conditions of less than 1% DMSO unless otherwise noted.

2.2. Inhibition of human neutrophil elastase Using the synthetic tetrapeptide substrate MeOSuc-AAPV-pNA (Bachem, Switzerland), the ability of the peptides of the invention to inhibit the hydrolytic activity of human neutrophil elastase (Serva Electrophoresis, Germany) It measured as follows.
The above substrate (0.3 mM) and human neutrophil elastase (10 nM) were incubated at 37 ° C. with a peptide (final concentration 1% DMSO) serially diluted with assay buffer (50 mM Tris, pH 8,300 mM NaCl, 0.01% Tween 20). Incubated at
The release of pNA was then followed by monitoring the change in absorbance at 405 nm for 30 minutes. Control assays without peptide under the same assay conditions described above proceeded linearly. Dose-response data was fitted to a four parameter Hill equation and IC ₅₀ values were determined using Graphpad (Prism 5).

2.3. Inhibition of porcine pancreatic elastase The ability of the peptides of the invention to inhibit the hydrolytic activity of porcine pancreatic elastase (Sigma, USA) was measured using the synthetic tripeptide substrate MeOSuc-AAA-pNA (Bachem, Switzerland) as follows. .
The substrate (1 mM) and porcine pancreatic elastase (15 nM) were combined at 37 ° C. with a peptide (final concentration 0.5% DMSO) serially diluted with assay buffer (50 mM Tris, pH 8, 100 mM NaCl, 0.01% Tween 20). Incubated.
The release of pNA was then followed by monitoring the change in absorbance at 405 nm for 30 minutes. Control assays without peptide under the same assay conditions described above proceeded linearly. Dose-response data was fitted to a four parameter Hill equation and IC ₅₀ values were determined using Graphpad (Prism 5).

2.4. Inhibition of human protease 3 Inactivation of human protease 3 (Elastin Products Company, USA) by the peptides of the present invention using the synthetic tripeptide substrate Boc-Ala-Ala-Nva-SBzl (Elastin Products Company, USA) It measured as follows.
A peptide obtained by serially diluting the substrate (1 mM), 4,4′-dithiodipyridine (250 μM) and human protease 3 (10 nM) with an assay buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.01% Tween 20) Incubated at 37 ° C. with a final concentration of 0.5% DMSO. The reaction process was followed by monitoring the change in absorbance at 340 nm for 30 minutes. Control assays without peptide under the same assay conditions described above proceeded linearly. Dose-response data was fitted to a four parameter Hill equation and IC ₅₀ values were determined using Graphpad (Prism 5).

3.0 Results The results of the experiments performed in 2.2 to 2.4 above are shown in Table 2 below.

Claims (12)

Formula cyclo ^{(-Xaa 1 -Xaa 2 -Thr 3 -Xaa} 4 -Ser 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Xaa 10 -Xaa 11 -Xaa 12 -Xaa 13 -)
(I)
[Where:
Xaa ¹ is OctGly, Arg, hArg, or Glu (phenethyl);
Xaa ² is Glu, Nle, hTyr, or Val;
Xaa ⁴ is Ala or AllylGly;
Xaa ⁶ is Ile;
Xaa ⁷ is Pro;
Xaa ⁸ is Pro;
Xaa ⁹ is Gln, or Tyr;
Xaa ¹⁰ is Lys;
Xaa ¹¹ is hLeu, Ser, Thr, Asn, Tyr, hGln, or His;
Xaa ¹² is ^D Pro, and
Xaa ¹³ is Pro;
However,
When Xaa ¹ is OctGly,
Xaa ² is Glu or Nle;
Xaa ⁴ is Ala;
Xaa ⁶ is Ile;
Xaa ¹⁰ is Lys;
Xaa ¹¹ is Ser, Thr, or Asn
Xaa ¹³ is Pro,
further
When Xaa ¹¹ is Tyr or His,
Xaa ¹ is Arg, hArg or Glu (phenethyl) ]
A cyclic skeleton peptide compound composed of 13 amino acid residues, and a pharmaceutically acceptable salt thereof. Xaa ¹ is OctGly, Arg, or Glu (phenethyl);
Xaa ² is Glu, Nle, hTyr, or Val;
Xaa ⁴ is Ala;
Xaa ⁶ is Ile;
Xaa ⁷ is Pro;
Xaa ⁸ is Pro;
Xaa ⁹ is Gln, or Tyr;
Xaa ¹⁰ is Lys;
Xaa ¹¹ is Ser, Thr, Asn, Tyr, or His;
Xaa ¹² is ^D Pro, and Xaa ¹³ is Pro;
However,
When Xaa ¹ is OctGly,
Xaa ² is Glu or Nle;
Furthermore, when Xaa ¹¹ is Tyr or His,
Xaa ¹ is Arg,
Claim 1 compounds of described. Xaa ¹ is Arg or hArg;
Xaa ² is Glu, Val, or hTyr;
Xaa ⁴ is Ala or AllylGly;
Xaa ⁶ is Ile;
Xaa ⁷ is Pro;
Xaa ⁸ is Pro;
Xaa ⁹ is Gln, or Tyr;
Xaa ¹⁰ is Lys;
Xaa ¹¹ is hLeu, Ser, Thr, or hGln;
Xaa ¹² is ^D Pro and Xaa ¹³ is Pro.
Claim 1 compounds of described. Cyclo (-OctGly-Glu-Thr-Ala -Ser-Ile-Pro-Pro-Gln-Lys-Asn- D Pro-Pro-);
Cyclo (-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Tyr-Lys-Thr- ^D Pro-Pro-);
Cyclo (-OctGly-Glu-Thr-Ala -Ser-Ile-Pro-Pro-Gln-Lys-Ser- D Pro-Pro-);
Cyclo (-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr- ^D Pro-Pro-);
Cyclo (-Arg-hTyr-Thr-Ala -Ser-Ile-Pro-Pro-Gln-Lys-Tyr- D Pro-Pro-);
Cyclo (-Arg-Nle-Thr-Ala -Ser-Ile-Pro-Pro-Gln-Lys-Tyr- D Pro-Pro-);
Cyclo (-Arg-Glu-Thr-AllylGly-Ser-Ile-Pro-Pro-Gln-Lys-Thr- ^D Pro-Pro-);
Cyclo (-Arg-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Tyr-Lys-Ser- ^D Pro-Pro-);
Cyclo (-hArg-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr- ^D Pro-Pro-);
Cyclo (-Arg-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-hLeu- ^D Pro-Pro-);
Cyclo (-Arg-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr- ^D Pro-Pro-);
Cyclo (-Arg-Glu-Thr-Ala -Ser-Ile-Pro-Pro-Tyr-Lys-hGln- D Pro-Pro-);
Cyclo (-Arg-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Tyr-Lys-Thr- ^D Pro-Pro-);
Cyclo (-Arg-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Tyr-Lys-His- ^D Pro-Pro-);
Cyclo (-Glu (phenethyl) -Glu-Thr-Ala-Ser -Ile-Pro-Pro-Gln-Lys-Tyr- D Pro-Pro-);
Cyclo (-Arg-Val-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr- ^D Pro-Pro-);
Cyclo (-Arg-hTyr-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr- ^D Pro-Pro-);
Cyclo (-Arg-Val-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Tyr- ^D Pro-Pro-)
Claim 1 Symbol placement compound selected from. For use as a medicament, free form or in the form of a pharmaceutically acceptable salt thereof, compound of according to any one of claims 1-4. Lung cancer, breast cancer, psoriasis, α1 antitrypsin deficiency, emphysema, cystic fibrosis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, bronchiectasis, pulmonary hypertension, pulmonary arterial hypertension, cardiac hypertrophy, myocarditis, acute myocardial infarction Treatment or prevention of rheumatoid arthritis, osteoarthritis, atherosclerosis, multiple sclerosis, pancreatitis, allergic rhinitis, systemic inflammatory response syndrome, inflammatory skin disease, inflammatory bowel disease, or Crohn's disease for, having inhibitory activity against elastase, free form or in the form of a pharmaceutically acceptable salt thereof, compound of according to any one of claims 1-4. In free form or pharmaceutically acceptable salt thereof, comprising mixtures and pharmaceutically inert carrier according to claim 1 of compounds according to any one of 4 or said compound, in particular inhalation, oral Suitable for topical, transdermal, injection, buccal, muscle, transmucosal, rectal, inhalation administration, especially tablets, dragees, capsules, solutions, liquids, gels, plasters, creams, ointments, syrups, slurries, suspensions, A pharmaceutical composition in the form of a powder or suppository. In particular, selective protease inhibitory activity and / or anti-cancer activity and / or anti-inflammatory activity and / or anti-infective activity and / or anti-cardiopathic activity and / or anti-immunological activity and / or anti-nerve activity against human neutrophil elastase as pharmaceutically active substances having a modified activity, free form or in the form of a pharmaceutically acceptable salt thereof, compound of according to any one of claims 1-4. Infection or a disease associated with such infection, or cancer ( such as lung cancer or breast cancer ) , or immunity mediated by elastase activity or caused by elastase activity, or mediated by elastase activity or caused by elastase activity Disease (such as psoriasis) or lung disease (such as alpha 1 antitrypsin deficiency, emphysema, cystic fibrosis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, bronchiectasis, pulmonary hypertension or pulmonary arterial hypertension), or Heart disease (such as cardiac hypertrophy, myocarditis or acute myocardial infarction), or neurodegenerative disease, or inflammation or inflammation-related disease (rheumatoid arthritis, osteoarthritis, atherosclerosis, multiple sclerosis, pancreatitis) , Allergic rhinitis, systemic inflammatory response syndrome, inflammatory skin disease, inflammatory bowel disease or Crohn's disease) Others for the prevention, as a medicament having an inhibitory activity against elastase, free form or in the form of a pharmaceutically acceptable salt thereof, according to claim 1 of any one of 4 compound or claim 7, wherein Composition . An infection or a disease associated with such infection, or cancer (such as lung cancer or breast cancer), or immunity mediated by elastase activity or caused by elastase activity, or mediated by elastase activity or caused by elastase activity Disease (such as psoriasis) or lung disease (such as alpha 1 antitrypsin deficiency, emphysema, cystic fibrosis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, bronchiectasis, pulmonary hypertension or pulmonary arterial hypertension), or Heart disease (such as cardiac hypertrophy, myocarditis or acute myocardial infarction), or neurodegenerative disease, or inflammation or inflammation-related disease (rheumatoid arthritis, osteoarthritis, atherosclerosis, multiple sclerosis, pancreatitis) , Allergic rhinitis, systemic inflammatory response syndrome, inflammatory skin disease, inflammatory bowel disease or Crohn's disease) Use of for the prevention, for the manufacture of a medicament having inhibitory activity against elastase, in the form of the free form or pharmaceutically acceptable salt thereof Compounds according to any of claims 1-4. An infection or such infection comprising a compound according to any of claims 1 to 4 , mediated by elastase activity or caused by elastase activity, or mediated by elastase activity or caused by elastase activity A pharmaceutical composition for the treatment of a disease or disorder associated with cancer, or cancer, or an immunological disease, lung disease, or heart disease, or neurodegenerative disease, or inflammation. Comprising the following steps, the production method (a) suitably functionalized solid support of compound according to any one of claims 1-4, the Xaa n ⁽ⁿ in the desired final product 13, 8,7,6,5 or 4) derivatives wherein the amino acid corresponding to N, is suitably N-protected (wherein any functional group present in the N-protected amino acid derivative is likewise suitably protected) Coupling with;
(B) removing the N-protecting group from the resulting product;
(C) The resulting product is a derivative in which the amino acid corresponding to Xaa ^n-1 in the desired final product is appropriately N-protected (wherein any functional group present in the N-protected amino acid derivative is As well as suitably protected);
(D) removing the N-protecting group from the product obtained in step (c);
(E) a derivative in which the amino acid at the n-2 position to the 1-position in the desired final product is appropriately N-protected (wherein any functional group present in the N-protected amino acid derivative is also appropriately protected) A step substantially corresponding to steps (c) and (d),
(F) if n is not 13, a derivative in which the amino acid at position 13 to n + 1 in the desired final product is appropriately N-protected (wherein any functional group present in the N-protected amino acid derivative) Is also suitably protected) and performs steps substantially corresponding to steps (c) and (d),
(G) detaching the resulting product from the solid support;
(H) cyclizing the product separated from the solid support;
(I) removing any protecting groups present on the functional group of the chain of amino acid residues, and (j) converting the resulting product to a pharmaceutically acceptable salt, if desired, or pharmaceutical The resulting salt, which is acceptable or unacceptable, is converted into the corresponding free form compound or a different pharmaceutically acceptable salt.